U.S. patent number 5,606,060 [Application Number 08/460,742] was granted by the patent office on 1997-02-25 for topoisomerase ii inhibitors and therapeutic uses therefor.
This patent grant is currently assigned to The United States of America as represented by the Department of Health. Invention is credited to Timothy L. MacDonald, Jose S. Madalengoitia, Yves Pommier.
United States Patent |
5,606,060 |
Pommier , et al. |
February 25, 1997 |
Topoisomerase II inhibitors and therapeutic uses therefor
Abstract
Azatoxin and derivatives thereof are illustrative of a new class
of antitumor drugs that are topoisomerase II (top 2) inhibitors.
The pharmacophore inhibits the catalytic activity of the purified
enzyme but does not unwind relaxed or supercoiled DNA. It is
nonintercalative and has at least two domains: a quasiplanar
polycyclic ring system, which may bind between DNA base pairs, and
a pendant substituent thought to interact with the enzyme, with the
DNA grooves or with both. In SV40 and c-myc DNA, azatoxin induces
numerous double-strand breaks according to a cleavage pattern which
differs from those of known top 2 inhibitors. Azatoxin also is a
potent inhibitor of tubulin polymerization.
Inventors: |
Pommier; Yves (Bethesda,
MD), MacDonald; Timothy L. (Charlottesville, VA),
Madalengoitia; Jose S. (Charlottesville, VA) |
Assignee: |
The United States of America as
represented by the Department of Health (Washington,
DC)
|
Family
ID: |
25510682 |
Appl.
No.: |
08/460,742 |
Filed: |
June 2, 1995 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
965922 |
Oct 23, 1992 |
|
|
|
|
868408 |
Apr 14, 1992 |
|
|
|
|
Current U.S.
Class: |
546/85; 546/86;
546/89 |
Current CPC
Class: |
C07D
471/04 (20130101); C07D 491/04 (20130101); C07D
499/14 (20130101) |
Current International
Class: |
C07D
471/00 (20060101); C07D 471/04 (20060101); C07D
491/00 (20060101); C07D 498/14 (20060101); C07D
491/04 (20060101); C07D 498/00 (20060101); C07D
498/04 (20060101); C07D 471/00 () |
Field of
Search: |
;546/86,92,80
;514/292,291 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
B H. Long, "Structure-Activity Relationships of Podophyllin
Congeners That Inhibit Topoisomerase II", DNA Topoisomerases in
Cancer Therapy, NCl Monographs, No. 4, 1987, pp. 123-127. .
Zwelling, et al., "Topoisomerase II as a Target of Antileukemic
Drugs", DNA Topoisomerases in Cancer Therapy, NCl Monographs, No.
4, 1987, pp. 79-82. .
Kohn, et al., "Topoisomerase II as a Target of Anticancer Drug
Action in Mammalian Cells", DNA Topoisomerases in Cancer Therapy,
NCl Monographs, No. 4, 1987, pp. 61-71. .
Chen, et al., "DNA Topoisomerases as Therapeutic Targets in Cancer
Chemotherapy", Chapter 4, Annual Reports in Medical Chemistry, vol.
21, 1986, pp. 257-262. .
Nelson, et al., "Mechanism of Antitumor Drug Action: Poisoning of
Mammalian DNA Topoisomerase II on DNA by
4'-(9-acridinylamino)-methanesulfon-m-anisidide", Proc. Natl. Acad.
Sci. vol. 81, 1984, pp. 1361-1365. .
Yang, et al., "Identification of DNA Topoisomerase II as an
Intracellular Target of Antitumor Epipodophyllotoxins in Simian
Virus 40-Infected Monkey Cells", Cancer Research, vol. 45, 1985,
pp. 5872-5876. .
L. F. Liu, "DNA Topoisomerase Poins as Antitumor Drugs", Annu. Rev.
Biochem., vol. 58, 1989, pp. 351-375. .
Macdonald, et al., "On the Mechanism of Interaction of DNA
Topoisomerase II with Chemotherapeutic Agents", DNA Topoisomerases
in Cancer, Chapter 16, 1991, pp. 199-214. .
M. R. Boyd, "Status of the NCl Preclinical Antitumor Drug Discovery
Screen", Principles & Practice of Oncology, vol. 3, No. 10,
1989, pp. 1-12. .
Monks, et al., "Feasibility of a High-Flux Anticancer Drug Screen
Using a Diverse Panel of Cultured Human Tumor Cell Lines", Journal
of the National Cancer Inst., Articles, vol. 83, No. 11, 1991, pp.
757-766. .
Paull, et al., "Display and Analysis of Patterns of Differential
Activity of Drugs Against Human Tumor Cell Lines: Development of
Mean Graph and COMPARE Algorithm", Journal of the National Cancer
Institute, REPORTS, vol. 81, No. 14, 1989, pp. 1089-1092. .
Chemical Abstracts Service CA113:152386 (Abstract of EP 357122).
.
Cancer Research, vol. 52 No. 16, Aug. 15, 1992, pp. 4478-4483, F.
Leteurtre et al. "Rational design and molecular effects of a new
topoisomerase II inhibitor azatoxin". .
Kawashima et al., Synthesis and pharmacological evaluation of
1,2,3,4-tetrahydro-beta-carboline derivatives, Chem. Pharm., Bull.,
43(5), 783-7 (Abstract). .
Lehnert et al., DNA topoisomerase II inhibition by substituted
1,2,3,4-tetrahydro-beta-carboline derivatives, Bioorg. Med. Chem.
Lett., 4(20), 2411-16 (abstract)..
|
Primary Examiner: Mai; Ngoclan
Assistant Examiner: Chi; Anthony R.
Attorney, Agent or Firm: Foley & Lardner
Parent Case Text
This application is a divisional of application Ser. No.
07/965,922, filed Oct. 23, 1992, which is a CIP of Ser. No.
07/868,408, filed Apr. 14, 1992, now abandoned.
Claims
What is claimed is:
1. A compound that inhibits topoisomerase II catalytic activity and
that is represented by the formula of compound: ##STR18## wherein
(i) R.sub.6 denotes F, Cl, Br, CN, OH, NH.sub.2 or H;
(ii) R.sub.5 denotes COOCH.sub.3, COCH.sub.3 or, COCH.sub.2 OH;
(iii) W and W' are the same or different and denote, respectively,
H or F; and
(iv) X denotes NH, S or O.
2. A compound according to claim 1, wherein R.sub.6 is F.
3. A compound according to claim 1, wherein R.sub.6 is Br.
4. A compound according to claim 1, wherein X is NH.
5. A pharmaceutical composition comprising a tumor-affecting amount
of a compound according to claim 1 and a physiologically compatible
carrier therefor.
6. A pharmaceutical composition according to claim 5, wherein said
composition is an injectable or infusible preparation.
7. A method of treating cancer in a mammal, comprising the step of
bringing a pharmaceutical composition according to claim 5 into
contact with cancerous tissue in a mammal that is suffering from a
tumor, such that neoplastic development in said cancerous tissue is
retarded or arrested.
Description
BACKGROUND OF THE INVENTION
Enzymes categorized under the rubric of "DNA topoisomerase" control
the topology of DNA over the course of conformational and
topological changes which occur during many cellular processes. For
example, DNA topoisomerases are involved in DNA replication, RNA
transcription and recombination.
Two kinds of DNA topoisomerases are recognized generally. Type I
and type II enzymes catalyze topological changes in DNA by
transiently breaking one stand or two strands of the DNA helix,
respectively. The relaxation of superhelical DNA is a
characteristic reaction catalyzed by a topoisomerase I ("top 1"),
while a topoisomerase II ("top 2") catalyzes the passing of two DNA
segments in a manner leading to such topoisomerization reactions of
DNA as supercoiling/relaxation, knotting/unknotting and
catenation/decantenation.
DNA topoisomerase II has been implicated as the chemotherapeutic
target for a diverse group of antitumor agents, including
epipodophyllotoxins, anthracyclines, acridines, anthracenediones
and ellipticines. See Macdonald et al., in DNA TOPOISOMERASES IN
CANCER 199-214 (Oxford University Press 1991) (hereafter "Macdonald
(1991)"), the contents of which are hereby incorporated by
reference. Under the influence of such drugs, top 2 is believed to
cleave DNA and form a concomitant covalent association with the
broken strand(s) of duplex DNA. The formation of such "cleavable
complexes" of drug, DNA and top 2 enzyme has been attributed to the
stabilization by the drug of a covalent, DNA-bound catalytic
intermediate in the cleavage-resealing sequence of the enzyme.
Id.
New inhibitors of top 2 have been identified after they were noted
for their antitumor properties. Some, such as amonafide, genistein
and the terpenoides, act in the manner of the above-mentioned
drugs, by trapping cleavable complexes. Antitumor compounds like
merbarone and fostriecin, by contrast, inhibit top 2 activity
without stabilizing cleavable complexes.
While attempting to elucidate mechanistic issues in this field,
including the nature of binding site(s) for top 2 inhibitors in the
ternary complex, Macdonald (1991) formulated a composite model by
superimposing structural subunits of top 2 inhibitors from the five
classes of compounds mentioned previously, namely,
epipodophyllotoxins, anthracyclines, acridines, anthracenediones
and ellipticines. The composite, three-domain pharmacophore
encompassed, inter alia, a family of hybrid structures which
incorporated, respectively, substructural elements from each class
of top 2 inhibitors. The "unified pharmacophore" model was not
refined sufficiently, however, to allow for a priori predictions of
any certainty regarding the activity, if any, of actual molecules
deemed within the ambit of the composite.
SUMMARY OF THE INVENTION
It is an object of the present invention, therefore, to provide top
2 inhibitors which display properties that not only distinguish
them from known inhibitor compounds but also recommend them for
therapeutic uses in anticancer and antiviral contexts.
In accomplishing this object and others there has been provided, in
accordance with one aspect of the present invention, compounds that
inhibit topoisomerase II catalytic activity and that are
represented by the following formulae (A)-(D): ##STR1## wherein (i)
Y represents the formula ##STR2## R1 and R3 are the same or
different and denote, respectively, hydrogen or methoxy, and
R2 denotes hydroxyl; and
(ii) R4 denotes hydrogen, hydroxyl, alkyl ether or hydroxyalkyl
ether;
In a preferred embodiment, R1 and R3 both denote methoxy and R4
denotes hydrogen; ##STR3## wherein (i) X denotes NH, S or O;
(ii) R5 denotes COOCH.sub.3, COCH.sub.3, COCH.sub.2 OH;
(iii) R.sub.6 denotes F, Cl, Br, CN, OH, H or NH.sub.2 ; and
(iv) W and W' are the same or different and denote, respectively, H
or F; ##STR4## wherein (i) X is the same as above;
(ii) W and W' are the same as above;
(iii) R.sub.6 is the same as above; and
(iv) R.sub.7 denotes H, OH, the formula ##STR5## wherein R denotes
H, OH F, Br, Cl, NO.sub.2, NH.sub.2, CN, OCH.sub.3, or CO.sub.2
CH.sub.2 CH.sub.3 ; the formula
wherein
T denotes NH or O;
Z denotes NH.sub.2, OH, N(CH.sub.3).sub.2, or N(CH.sub.2 CH.sub.2
Cl);
n is 2, 3 or 4; and
R.sub.7 may be derivatized with a 4,6 o-protected sugar; ##STR6##
wherein (i) X is the same as above;
(ii) W and W' are the same as above;
(iii) R.sub.6 is the same as above;
(iv) R.sub.7 is the same as above; and
(v) R.sub.8 denotes H or OH.
The term "4,6 o-protected sugar" includes etoposide analogs such as
o-glucosyl sugars protected with a conventional protecting group
such as a methy acetal or a thiophene acetal group.
It has surprisingly been found that compounds of formula (C), when
R.sub.6 is H, R.sub.7 is ##STR7## and R is F, have increased
topoisomerase II catalytic activity when compared to compounds of
formula (A). The activity is increased by a factor of at least 5,
and preferably by at least 10, when compared to compounds of
formula (A). Those skilled in the art readily recognize that the
dosage amount required for compounds having such an increase in
activity are respectively decreased to maintain the same or a
similar effect.
In accordance with another aspect of the present invention, a
pharmaceutical composition is provided that comprises a
tumor-affecting amount of a compound represented by formula (A),
(B), (C) or (D), and a physiologically compatible carrier for that
compound. In one preferred embodiment, the pharmaceutical
composition is an injectable or infusible preparation.
In accordance with yet another aspect of the present invention, a
method is provided for treating cancer in a mammal, which method
comprises the step of bringing a pharmaceutical composition as
described above into contact with cancerous tissue in a mammal
suffering from a tumor, such that neoplastic development in the
cancerous tissue is retarded or arrested. Thus, preferred modes of
administration in the context of such a method are those that
maximize contact between cancerous tissues and the active agent of
the pharmaceutical composition.
Other objects, features and advantages of the present invention
will become apparent from the following detailed description. It
should be understood, however, that the detailed description and
the specific examples, while indicating preferred embodiments of
the invention, are given by way of illustration only, since various
changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graphical representation of results generated by
testing the inhibitory activity of an illustrative compound within
the present invention against panel of tumor cell lines which is a
standard of the National Cancer Institute Developmental
Therapeutics Program [GI50: "50% growth inhibition"; TGI: "tumor
growth inhibition"; LC50: "50% lethal concentration"].
FIG. 2 depicts, at left, the structural formula of a preferred
compound of the present invention (azatoxin) and, at right, a
stereochemical superimposition of the top 2 poison
demethyldesoxy-podophyllotoxin (DMDP; dashed lines) and azatoxin
(solid lines).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
A new class of top 2 poisons has been discovered which, despite
certain structural similarities to demethylepipodophyllotoxins and
other known top 2 inhibitors, are distinguishable in terms of DNA
cleavage pattern and structure-activity relationships which could
not have been predicted from any structural superimposition of
known anthracycline, acridine, epipodophyllotoxin and
anthracenedione structures. Archetypical of the new class of
inhibitors is the compound azatoxin and derivatives thereof,
5R,11aS-3-one-1H,6H,-5,4,11,11a-tetrahydro-5-(3,5-dimethoxy-4-hydroxypheny
l)-oxazolo[3',4':1,6]pyrido[3,4-b]indole, which is represented by
formula (A) above when R1 and R3 are methoxy, R2 is hydroxyl and R4
is hydrogen.
Compared to other top 2 inhibitors, azatoxin induces the largest
number of top 2 cleavage sites both in SV40 and c-myc DNA, and is
very active in inducing protein-linked DNA breaks in cells.
Accordingly, azatoxin and other pharmacophores of the present
invention should be useful as reagents in the context of mapping
top 2 sites in chromatin.
Azatoxin also displays a pattern of differential growth inhibition
against human tumor cell lines which is indicative of an antitumor
drug action reminiscent of that of top 2 poisons like the
nonintercalative epipodophyllotoxins, VP-16 (etoposide) and VM-26
(teniposide). See Yang et al., Cancer Res. 45: 5872-76 (1985), and
Liu (1989), supra, at 361-63. More specifically, azatoxin evidenced
significant inhibitory activity when screened against a panel of
sixty human tumor cells lines representing leukemia and melanoma,
as well as cancers of the lung, colon, kidney, ovary and central
nervous system. Pursuant to the convention of Paull et al., J.
Nat'l Cancer Inst. 81: 1088-92 (1989), the contents of which are
hereby incorporated by reference, FIG. 1 depicts these results, in
terms of parameters conventional to cancer research, by graphs
which are centered at the arithmetic mean of the logarithm of each
parameter. See also Monks et al., J. Nat'l Cancer Inst. 83: 757-66
(1991), and Boyd, Principles & Practice of Oncology 3: 1-12
(1989).
Since a right-extending bar in such a "means graph" indicates a
sensitivity by the cell line to the test substance, FIG. 1
evidences the cytotoxicity of aratoxin to cells associated with
disseminated cancers (leukemias) as well as several consolidated
cancers (non-small cell lung and colon). It also has been
discovered, using a conventional in vitro tubulin polymerization
assay, that azatoxin is a potent tubulin inhibitor in the manner of
several compounds, such as vineristine, vinblastine and taxol, that
are very active in cancer chemotherapy. Thus, azatoxin effectively
prevents tubulin polymerization in vitro at concentrations in the
range of 1 to 10 .mu.M.
Compounds of the present invention can be synthesized via a
modified Pictet-Spengler reaction. More specifically, the inventive
compounds are obtained by reacting a corresponding pendent-group
dimethylacetal with a precursor carbamate in the presence of a
catalytic amount of para-toluenesulfonic acid.
Because the azatoxin skeleton is synthetically accessible, a large
number of derivatives are readily prepared and screened for top 2
inhibitory activity in accordance with the present invention. Such
screening can be effected by means of an in vitro assay which
employs purified top 2 and .sup.32 P-end-labelled DNA. In such an
assay, top 2 inhibition by a test substance results DNA
fragmentation which can be detected by agarose gel electrophoresis,
as described by Fesen and Pommier, J. Biol. Chem. 19: 11354-59
(1989); by the filter-binding assay described by Pommier et al.,
Biochem. 24: 6410-16 (1985); or by the sodium dodecylsulfate
precipitation assay employed by Nelson et al., Proc. Nat'l Acad.
Sci. USA 81: 1361-65 (1984).
Although a myriad of azatoxin derivatives may be synthesized, the
structure-activity relationships illuminated in this description
indicate that those derivatives falling within the present
invention will conform to certain guidelines of molecular design.
Thus, with reference to formula (A) and FIG. 1, there should be a
conservation of the relative spatial orientation between the
polycyclic ring system and the pendant ring (Y) in azatoxin. The
R/S stereochemistry of the polycyclic ring system also should be
carried over from azatoxin. Methoxy groups at the 3' and 5'
positions (R1 and R3, respectively) enhance top 2 inhibition, while
a 4' hydroxyl group (R2) is essential for inhibitory activity. In
contrast to R2, greater flexibility is realized at the 11R position
(R4), where substitutions of the preferred hydrogen can be a
hydroxyl group, an alkyl ether group or a hydroxyalkyl ether group
such as ethoxyethyl and hydroxypropyl.
The present invention also contemplates the use of compounds
according to formulae (A), (B), (C) and (D) in a pharmaceutical
composition which further comprises a physiological compatible
carrier for the compound. The phrase "physiological compatible
carrier" here denotes a solid, liquid or gaseous material that can
be used as a vehicle for administering a formula (A), (B), (C), or
(D) compound as a medicament because the material is inert or
otherwise medically acceptable, as well as compatible with the
active agent, in a particular context of administration. In
addition to a suitable excipient, a physiologically compatible
carrier can contain conventional additives such as diluents,
adjuvants, antioxidants and other preservatives, solubilizing
agents, and the like.
The preferred routes for administering a pharmaceutical composition
of the present invention are those that maximize introduction of
the active agent into the immediate region of the tumor under
treatment. It is advantageous, therefore, that the pharmaceutical
composition should be an injectable or infusible preparation, the
formulation of which would typically require initial solubilization
in a nonaqueous solvent, such as dimethyl sulfoxide (DMSO), that is
employed in the field to accommodate hydrophobic anticancer agents.
Similarly, intraarterial administration is preferred for therapy
when the tumor is supplied by a known artery, while intrathecal
administration can be used for tumors located in the brain.
Intradermal and intracavitary administration is feasible with
tumors that are restricted to areas close to a particular skin
region and particular portion of the body cavity, respectively. By
the same token, an active agent of the present invention can be
administered via application to a mucosal lining (sublingually, for
example), when the tumor is accessible through the lining, or via
inhalation or insufflation with a gaseous carrier, when the tumor
is accessible to the respiratory tract. It is possible,
furthermore, to administer an active agent of the present invention
in a topical preparation applied to a superficial tumor or, more
generally, to a lesion associated with a viral infection against
which compounds of the present invention prove effective.
Alternatively, an inventive pharmaceutical preparation can be in a
form suitable for oral administration (cachet, tablet, hard- and
soft-gelatin capsule, etc.). This route takes advantage of the
targeting afforded by the particular sensitivity of proliferating,
neoplastic cells to the cytoxic effects of the inventive
compounds.
A pharmaceutical composition of the present invention also can take
the form of a solid dosage preparation (implant) for introduction
surgically into the tumor or its immediate vicinity. A so-called
"implantation tablet" would be made up primarily of the active
substance and, hence, could be absorbed completely. On the other
hand, an implant featuring a non-absorbable skeleton (plastic
matrix) or coating would effect controlled release of the inventive
compounds upon implantation and then would be removed from the
tissues after ceasing to exert an antitumor influence.
A pharmaceutical composition within the present invention
preferably contains an inventive compound in an amount that itself
is tumor-inhibiting or, in the context an infusion regimen, that
permits accumulation in the tumor of a cytotoxic level of the
active agent. Since an inventive compound typically inhibits both
topoisomerase II activity and tubulin polymerization, it is
possible to realize a therapeutic spectrum combining aspects both
of a top 2 poison like doxorubicin and a tubulin inhibitor like
vincristine. See GOODMAN AND GILLMAN'S THE PHARMACOLOGICAL BASIS OF
THERAPEUTICS (7th ed. 1985), at pages 1279-85, the contents of
which are hereby incorporated by reference. Symptoms of clinical
toxicity associated with these two types of anticancer agents also
may be observed and, if so, will require remedial countermeasures
which are conventional to the field of cancer therapy.
From these considerations it will be apparent that the optimum
dosage of the inventive compound will vary with the particular
case. The relevant pharmacokinetics can be determined routinely in
the clinical context, which may be therapeutic or prophylactic.
"Therapeutic treatment" in this context means treatment intended to
kill a maximum number of neoplastic cells, while a "prophylactic
treatment" is one aimed at retarding or preventing re-establishment
of a proliferating neoplastic population and, hence, a relapse in
the disease, once remission has been achieved. It is anticipated
that a typical dosage regimen will be similar to that of etoposide
(VP-16), i.e., on the order of 100 mg/m.sup.2 per day.
Without further elaboration, it is believed that those skilled in
the art, informed by the preceding description, can utilize the
present invention fully. Accordingly, the following examples are
presented for purposes of illustration only. The materials and
methods employed in the examples are described below:
Chemicals, and enzymes: Azatoxin and its derivatives, as well as
the demethyldesoxypodophyllotoxin (DMDP) and
demethylepipodophyllotoxin (DMEP), were synthesized by conventional
methods. The compounds m-AMSA and mitoxantrone were obtained from
the Drug Synthesis and Chemistry Branch, National Cancer Institute,
Bethesda, Md. The compounds VP-16 and VM-26 were obtained from
Bristol-Myers Company (Wallingford, Conn.). Drug stock solutions
were made in dimethylsulfoxide at 10 mM. Further dilutions were
made in distilled water. Various other reagents, including simian
virus 40 (SV40) and c-myc human DNA inserts in plasmid pBR322,
restriction endonucleases, DNA topoisomerase I, T4 polynucleotide
kinase, calf intestine phosphatase, agarose and polyacrylamide/bis,
were purchased from Bethesda Research Laboratories (Gaithersburg,
Md.), from the American Type Culture Collection (Rockville, Md.) or
from New England Biolabs (Beverly, Mass.). [Gamma-.sup.32 P]ATP was
purchased from New England Nuclear Research Products (Boston,
Mass.).
DNA topoisomerase II was purified from mouse leukemia L1210 cell
nuclei as described, for example, by Pommier et al. (1985), supra,
and was stored at -70.degree. C. in 40% (v/v) glycerol, 0.35M NaCl,
5 mM MgCl.sub.2, 1 mM KH.sub.2 PO.sub.4, 0.2 mM dithiothreitol and
0.1 mM phenylmethanesulfonyl fluoride (pH 6.4). The purified enzyme
yielded a single 170 kDa band after silver staining of
SDS-polyacrylamide gels, in accordance with the description of
Pommier et al., Cancer Res. 46: 3075-81 (1986).
Preparation of end-labeled DNA fragments: DNA fragments were 5'
end-labeled as described, for example, by Fesen and Pommier (1989),
supra. Briefly, native DNA was first linearized with a restriction
enzyme, then the 5'-DNA was first linearized with a restriction
enzyme, then the 5'-DNA termini were dephosphorylated with calf
alkaline phosphatase and labeled with [gamma-.sup.32 P]ATP using T4
polynucleotide kinase. For double-strand breaks assays using HL-60
nuclear extract, SV40 DNA was digested with BclI endonuclease and
labeled at both DNA termini. For sequencing experiments, SV40 and
c-myc DNAs were first 5'-end labeled at the XhoII and XbaI
restriction sites, respectively. Then, in order to generate
uniquely 5'-end-labeled fragments, labeled DNA was subjected to a
second enzyme digestion, PflMI for SV40, and EcoRI plus HindIII for
c-myc DNA. The resulting DNA fragments were separated by agarose
gel electrophoresis and isolated by electroelution. Purification by
phenol-chloroform extraction and ethanol precipitation were
included between each step and at the end of the labeling
procedures, pursuant to Pommier et al., J. Molec. Biol. 222: 909-24
(1991).
Topoisomerase II-induced DNA cleavage reactions: DNA fragments were
equilibrated with or without drug in 10 mM Tris-HCl, pH 7.5, 50 mM
KCl, 5 mM MgCl.sub.2 0.1mM EDTA, 1 mM ATP and 15 .mu.g/ml bovine
serum albumin for 5 minutes before addition of purified
topoisomerase II (40 to 70 ng) or HL-60 nuclear extract in 20 .mu.l
final reaction volume. Reactions were stopped by adding sodium
dodecyl sulfate (SDS) to a final concentration of 1% and proteinase
K to 400 .mu.g/ml, followed by incubation for 1 hour at 40.degree.
C.
For agarose gel analysis, 3 .mu.l (10.times.) loading buffer (0.3%
bromophenol blue, 16% Ficoll, 10 mM Na.sub.2 HPO.sub.4) was added
to each sample which was then heated at 65.degree. C. for 1-2
minutes before loading into an agarose gel made in (1.times.) TBE
(89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH8), in accordance with
Fesen & Pommier (1989), supra. Agarose gel electrophoresis was
conducted overnight at 2 V/cm. The quantification of drug-induced
DNA double-strand breaks in the presence of HL-60 nuclear extract
was effected as described in the next paragraph.
Radioactive gels were counted in betascope 603 blot analyzer. For
each lane radioactivity then was measured in the DNA cleavage
products (C) (size between 600-5243 bp), and in the total DNA
present in the lane with a size superior to 600 bp (T).
Drug-induced cleavage was expressed as: ##EQU1## where C. and T.
are the counts for cleaved and total DNA, respectively, in presence
of nuclear extract without drug.
For DNA sequence analysis, samples were precipitated with ethanol
and resuspended in 2.5 .mu.l loading buffer (80% formamide, 10 mM
NaOH, 1 mM EDTA, 0.1% xylene cyanol and 0.1% bromophenol blue).
Samples were heated to 90.degree. C. and immediately loaded into
DNA sequencing gels (6% polyacrylamide; 19:1, acrylamide:bis)
containing 7M urea in (1.times.) TBE buffer. Electrophoresis was at
2500 V (60 W) for 4 hours. Gels were dried on 3M paper sheets and
autoradiographed with Kodak XAR-2 film. See Pommier et al. (1991),
supra.
EXAMPLE 1
Synthesis of Azatoxin and Other Compounds
1H NMR spectra were taken on a General Electric QE300 Spectrometer
at 300 MHz. Mass spectra were recorded on a Finnegan MAT4615
GC/MS/DS instrument using chemical or electron impact ionization
techniques. Elemental analyses were determined by Atlantic Microlab
Inc. (Norcross, Ga.). Melting points were determined on a
Thomas-Hoover UNI-MELT apparatus and are uncorrected.
Thin-layer chromatography was performed using E. Merck glass plates
pre-coated with silica gel 60 F-254 and visualized with a
phosphomolybdic acid/ethanol solution. Woelm silica 32-63 was
employed for column chromatography which was carried out using a
modified short/flash column technique.
Tetrahydrofuran was distilled from sodium benzophenone immediately
prior to use. Dichloromethane was distilled from CaH.sub.2
immediately before use. All chemicals were purchased from Aldrich
Chemical Company except for D and L-tryptophan methyl ester-HCl,
which was purchased from Sigma Chemical Company.
All reactions were carried under an argon atmosphere.
4S-(1H-Indol-3-ylmethyl)-2-oxazolidone (Compound 1A):
Sodium (1.56 g, 68.1 mmol) was dissolved in absolute ethanol (150
ml) and 1-trptophanol (12.94 g, 68.02 mmol) in ethanol (100 ml) and
diethyl carbonate (8.83 g, 74.8 mmol) were added. The solution was
heated at reflux for 5 hours and was concentrated after cooling.
Saturated NH.sub.3 Cl (100 ml) and CH.sub.2 Cl.sub.2 (200 ml) were
added, and after mixing well the layers were separated. The organic
layer was washed with CH.sub.2 Cl.sub.2 (2.times.100 ml) and the
organic fractions were combined, dried over Na.sub.2 SO.sub.4, and
concentrated. Recrystallization from MeOH/H.sub.2 O yielded 11.66 g
(79%) of a white solid: mp 155.sub.0 C; .sub.1 NMR (CDCL.sub.3)
8.17(sb, 1H) 7.57(d, J=7.94 Hz, 1H), 7.40(d, J=8.06 Hz, 1H),
7.19(m, 2H), 7.08(d, J=2.2 Hz, 1H), 5.22(sb, 1H), 4.50(m, 1H),
4.21(m, 2H), 3.04(m, 2H). [g].sup.24.sub.D +8.4 (c 9.4, MeOH).
Anal. (C.sub.12 H.sub.12 N.sub.2 O.sub.2) C, H, N.
4R-(1H-Indol-3-ylmethyl)-2-oxazolidone:
This compound was prepared in a manner analogous to the preparation
of Compound 1a. [a].sup.24.sub.D -8.3 (c 1.25, MeOH), Anal.
(C.sub.12 H.sub.12 N.sub.2 O.sub.2) C, H, N.
General Method far the Preparation of Dimethyl Acetals
To a solution of the aromatic aldehydes (1 g) in trimethyl
orthoformate (7 ml) a catalytic amount of p-TSOH (40 mg) was added
and the reaction was followed to completion by TLC. The solvent was
removed under reduced pressure and the remaining oil was dissolved
in CHCl.sub.2 and filtered through a plug of silica. The solvent
was again removed under reduced pressure and the remaining oil was
stored in a desiccator until use.
Preparation of 5,11a-trans-aza toxins:
Method A
To a solution of Compound 1a (2 mmol) and the corresponding
aldehyde (2 mmol) in a CH.sub.2 Cl.sub.2 /MeOH (9:1) solution (6
ml) was added concentrated H.sub.2 SO.sub.4 (4 mmol). The reaction
was followed to completion by TLC (20% acetone in CHCL.sub.3). The
solution was added to sat NaHCO.sub.3, the layers were separated,
and the aqueous layer was washed with CH.sub.2 Cl.sub.2 (3.times.).
The combined organic fractions were dried over Na.sub.2 SO.sub.4,
filtered and concentrated. The product was purified by flash
chromatography (acetone-CH.sub.2 Cl.sub.2).
Method B
To a solution of the corresponding dimethyl acetal (2.2 mmol) and
Compound 1a (2 mol) in CHCl.sub.3 (8 ml), p-toluenesulfonic acid
(0.2 mmol) was added. The reaction was followed to completion by
TLC. If the reaction proceeded too slowly the reaction was heated
at reflux. Saturated NaHCO.sub.3 was added. The layers were
separated, and the aqueous layer was washed with CH.sub.2 Cl.sub.3
(2.times.20 ML). The organic layers were combined, dried over
Na.sub.2 SO.sub.4, filtered and concentrated. The product was
purified by flash chromatography (acetone-CH.sub.2 Cl.sub.2).
Method C
To a solution of the corresponding dimethyl acetal (3 mmol) and
Compound 1a (2 mmol) in anhydrous THF (8 ml), anhydrous TFA (10
mmol) was added and the solution was heated at reflux. The reaction
was followed to completion by TLC. After cooling, the solution was
added to saturated NaHCO.sub.3, the layers were separated, and the
aqueous layer was washed with CH.sub.2 Cl.sub.2 (3.times.20 ml).
The combined organic fractions were dried over Na.sub.2 SO.sub.4,
filtered and concentrated. The product was purified by flash
chromatography (acetone-CH.sub.2 Cl.sub.2).
5R,11aS-3-One-5,4,11,11a-tetrahydro-5-(3,5-dimethoxy-4-hydroxyphenyl)-1H,6H
-oxazolo[3',4':1,6]pyrido[3,4-b]indole (Compound 1)
This compound was prepared as described in method C. Purification
by flash chromatography (12% acetone in CHCl.sub.3, R.sub.f =0.28)
produced a white solid in 91% yield: mp (decomposed slowly around
175.degree. C.); .sub.1 NMR (CD.sub.3 CN) 7.94(sb, 1H), 7.51(d,
J-7.91 Hz, 1H), 7.30(d, J=7.57 Hz, 1H), 7.09(m, 2H), 6.59(s, 2H),
6.27(s, 1H), 5.88(d, J=1.7 Hz, 1H), 4.54(dd app t, J=8.3 Hz, 1H),
4.33(m, 1H), 4.21(dd, J=8.5 Hz, 4.7 Hz, 1H), 3.75(s, 6H), 3.16,(dd,
J=15 Hz, 4.6 Hz, 1H), 2.76(ddd, J=15 Hz, 10.38 Hz, 1.73 Hz, J=1.73
Hz, 1H) [.alpha.].sup.22.sub.D -139.6. Anal
(C.sub.21,H.sub.20,N.sub.2,O.sub.5) C, H, N.
5S,11aR-3-One-5,4,11,11a-tetrahydro-5-(3,5-dimethoxy-4-hydroxyphenyl)-1H,6H
-oxazolo[3,4,:1,6]pyrido[3,4-b]indole
This compound was prepared in a manner analogous to the preparation
of Compound 1. [.alpha.].sup.22.sub.D =+139.4. Anal.
(C.sub.21,H.sub.20,N.sub.2,O.sub.5) O, H, N.
5R,11aS-3-One-5,4,11,11a-tetrahydro-5-(3-methoxy-4-hydroxyphenyl)-1H,6H-oxa
zolo[3',4':1.6]pyrido[3,4-b]indole (Compound 2)
This compound was prepared as described in method A. Purification
by flash chromatography (15% acetone in CHCl.sub.3, R.sub.f =0.30)
gave a white solid in 40% yield: .sup.1 NMR (CD.sub.3 CN) 8.94(s,
1H), 7.51(d, J=7.59 Hz, 1H), 7.29(d, J=7.95 Hz, 1H), 7.09(m, 2H),
6.91(d, J=1.7 Hz, 1H,), 6.79(d, J=8.08 Hz, 1H), 6.74(dd, J=8.08 Hz,
1.7 Hz 1H), 6.55(sb, 1H), 5.90(d, J=1.5 Hz, 1H), 4.52(dd app t,
J=7.94 Hz, 1H), 5.27(m.1H), 4.20(dd, J=8.21 Hz, 4.83 Hz, 1H),
3.78(s, 3H), 3.16(dd, J=14.93 Hz, 8.48 Hz, 1H), 3.77(ddd, J=14.91
Hz, 10.07 Hz, 1.59 Hz, 1H). Anal. (C.sub.20 H.sub.18 N.sub.2
O.sub.4) C, H, N.
5R,11aS-3-one-5,4,11,11a-tetrahydro-5-(4-hydroxyphenyl)-1H,6H-oxazolo[3',4'
:1,6]pyrido[3,4-b]indole (Compound 3)
This compound was prepared as described in method C. Purification
by flash chromatography (20% acetone in CHCl.sub.3, R.sub.f =0.28)
followed by recrystallization from CH.sub.3 CN gave a white solid
in 89% yield: .sub.1 NMR (d.sub.6 -DMSO) 9.47(s, 1H), 7.43(d,
J=7.56 Hz, 1H), 7.24(d, J=7.91 Hz, 1H), 6.99(m, 4H), 6.70(d, J=8.6
Hz, 2H), 5.81(s, 1H), 4.49(dd app t, J=8.03 Hz, 1H), 4.14(m, 2H),
3.10(dd, J=12.14 Hz, 4.69 Hz, 1H), 2.68(dd, J=14.3 Hz, 10.6 Hz,
1H). Anal. (C.sub.19,H.sub.16,N.sub.2,O.sub.3 /CH.sub.3 CN) C, H,
N.
5R,11aS-3-one-5,4,11,11a,tetrahydro-5-(3,4,5-trimethoxyphenyl)-1H,6H-oxazol
o[3',4':1,6]pyrido[3,4-b]indole (Compound 4)
This compound was prepared as described in method C. Purification
by flash chromatography (7% acetone in CHCl.sub.3, R.sub.f =0.30)
yielded a white solid in 91% yield: mp 203.degree. C., .sup.1 NMR
(CD.sub.3 CN) 8.95(s, 1H), 7.51(d, J=7.64 Hz, 1H), 7.31(d, J=7.93
Hz, 1H), 7.09(m, 2H), 6.61(s, 2H), 5.89(s, 1H), 4.57(dd app t,
J=8.24 Hz, 1H), 4.35(m, 1H), 4.23(dd, J=8.42 Hz, 4.68 Hz, 1H),
3.74(s, 6H), 3.70(s, 3H), 3.17(dd, J=15.02 Hz, 4.48, Hz, 1H),
2.77(dd, J=14.85 Hz, 10.41 Hz, 1H).
Methyl-3-(1-benzenesulfonyl-indol-2-yl)-2-aminopropanoate (Compound
8a)
This compound was prepared, according to the method of Schollkopf
et al., Angew, Chem. Int. Ed. Engl. 18: 863 (1979), using
(1-benzenesulfonyl)-2-chloromethyl-indole (Compound X) and
2,5-diethoxy-3,6-tetrahydropyrazine (Compound Y) as starting
materials. 'H NMR(CDCl.sub.3) 8,19(d,1H), 7,79(d, 2H) ,
7,55-7,18(m, 6H), 6.51(s,1H), 4,19(q, 2H), 4,00(dd, 1H), 3.55(dd,
1H), 1.18(t, 3H).
3-(1-benzenesulfonyl-indol-2-yl)-2-aminopropanol (Compound 8b)
To a well-stirred solution of 0.44 g (4.1 eq.) NaBH.sub.4 in 20 mL
75% ethanol, 0.85 g (8a) in 10 mL 75% ethanol was added and the
solution heated at reflux 15 hours. TLC showed no starting
material. The solution was allowed to cool and then it was diluted
with 20 mL water and the ethanol was removed by rotary evaporation.
The residue was extracted with ethyl acetate (4.times.20 mL), dried
over sodium sulfate, and concentrated to yield 0.56 g (56%) of
Compound 8b as a clear oil which was used without further
purification. 'H NMR (CDCl.sub.3) 8.18(d, 1H), 7.71(d, 2H),
7.60-7.19(m, 6H), 6.50(s, 1H), 3.70(m, 1H), 3.50(dd, 1H), 3.45(dd,
1H), 3.25(dd, 1H), 2.90(dd, 1H).
1H-Indol-2-ylmethyl-2-oxazolidone (Compound 8c)
To a solution of 0.07 g (1.5 eq) Na dissolved in 5 mL absolute
ethanol, 0.67 g of Compound 8b in 10 mL absolute ethanol and 0.36 g
(1.5 eq) diethyl carbonate were added and the solution was heated
at reflux overnight (16 hours). TLC showed a higher R.sub.f
spot(80:20) ethyl acetate: hexane, R.sub.f =0.35). After cooling
the ethanol was evaporated by rotary evaporation and the residue
was diluted with 15 mL saturated ammonium chloride and extracted
with CH.sub.2 Cl.sub.2 (3.times.20 mL). Purification by flash
chromatography using an 80:20 ethyl acetate:hexane mixture as
eluent yielded 0.22 g (71%) of Compound 8c as a tan solid. 'H
NMR(CDCl.sub.3) 8.85(s, 1H), 7.55(d, 1H), 7.30(d, 1H), 7.15(m, 2H),
6.45(s, 1H), 6.20(g, 1H), 4.30(m, 1H), 4.00(m, 2H), 2.82(d,
2H).
5,11a-trans-3-one-5,4,11,11a-tetrahydro-5-(3,5-dimethoxy-4-hydroxyphenyl-1H
,10H-oxazolo[3',4':1,6]pyrido[4,3-b]indole (Compound 8)
To a solution of 0.7 g (1.5 eq) syringealdehyde dimethyl acetal and
a few grains p-TSOH in 2 mL CH.sub.2 Cl.sub.2 was added 0.04 g of
Compound 8c. The solution was stirred for 2 hours. TLC showed no
starting material. The solution was concentrated and purified by
flash chromatography using a 15% acetone in chloroform solution as
eluent to yield 0.05 g (69%) of Compound 8 as a beige solid. 'H
NMR(CDCN) 9.28(bs, 1H), 7.40(d, 1H), 7.15(t, 1H), 7.00(d, 1H),
6.92(t, 1H), 6.58(s, 2H), 6.22(bs, 1H), 5.98(s, 1H), 4.47(t, 1H),
4.31(m, 1H), 4.16(q, 1h), 3.69(s, 6H), 3.11(dd, 1H), 2.91(ddd,
1H).
Racemic methyl-3-(acenapth-1-yl)-2-aminopropanoate (Compound
7a)
This compound was synthesized according to the method of Schollkopf
et al., Liebigs Ann. Chem. 1987: 393-97, using
acenapthylene-1-carboxaldehyde (Compound Z) and
2,5-diethoxy-3,6-tetrahydropyrazine (Compound Y) as starting
materials. The final product was synthesized in an identical manner
as described for Compound 8. Due to the substitution in the
4-position, however, the intermediates existed as an inseparable
mixture of diastereomers and 'H NMR analysis proved impossible,
except for identification of important groups.
5,11a-trans-3-one-5,4,11,11a-tetrahydro-5-(3,5-dimethoxy-4-hydroxyphenyl)-1
H, 10H-oxazol[3',4':1,6]pyrido[1,2-b]acenapthylene (Compound 7)
0.01 mL BP.sub.3 OEt.sub.2 was added to a solution of 0.03 g of
Compound 7d and 0.01 mL triethyl silane in 1 mL CH.sub.2 Cl.sub.2
at -78.degree. C. The solution was warmed slowly to room
temperature over a period of 2 hours. TLC showed no starting
material. 5 mL H.sub.2 O was added, the layers were separated, and
the aqueous layers were extracted with 2.times.5 mL portions of
CH.sub.2 Cl.sub.2, dried and concentrated. The residue was purified
by flash chromatography using a 15% acetone in chloroform solution
as eluent to yield 19.4 mg (70%) of Compound 7. 'H NMR(CDCl.sub.3)
7.81(d, 1H), 7.71(d, 1H), 7.65-7.51(m, 2H), 7.38(t, 1H"), 7.11(d,
1H), 6.72(s, 2H), 6.14(d, 1H), 5.52(s, 1H), 4.58(t, 1H), 4.25(m,
2H), 3.77(s, 6H), 3.24(dd, 1H), 2.90(ddd, 1H).
1R,3R-1-(3-5-dimethoxy-4-hydroxyphenyl)-3-methoxycarbonyl-1,2,3,4-tetrahydr
o-.beta.-carboline (Compound 9A)
To a solution of 1-tryptophan methyl ester hydrochloride (9.52 g,
37.4 mmol) in CHCl.sub.3 (150 ml) was added 14% ammonium hydroxide
(30 ml). The biphasic mixture was allowed to stir for 1 hour. The
layers were separated and the aqueous layer was extracted with
CHCl.sub.3 (2.times.100 ml). The organic layers were combined,
dried over Na.sub.2 SO.sub.4, and concentrated to yield a yellow
oil. The oil was dissolved in benzene (200 ml). Syringaldehyde
(6.81 g, 37.4 mmol) and Na.sub.2 SO.sub.4 (10 g) were added, and
the solution was allowed to stir for 60 hours. A white precipitate
formed. The mixture was again concentrated, and anhydrous CH.sub.2
Cl.sub.2 (150 ml) and anhydrous TFA (5.76 ml, 74.8 mmol) were added
at 0.degree. C. The solution was allowed to stir at 0.degree. C.
for 12 hours. The mixture was again concentrated, and the remaining
solid was added to a biphasic mixture of saturated NaHCO.sub.3 and
ether. The mixture was allowed to stir for 1.5 hours and the white
solid that formed was collected in a sintered glass funnel. The
solid was washed with water, dried in a vacuum oven, and
recrystallized from CH.sub.3 CN/water to produce 13.34 g (93%) of a
white solid: Anal. (C.sub.21,H.sub.22,N.sub.2,O.sub.3) C, H, N.
1S,3S-1-(3,5-dimethoxy-4-hydroxyphenyl)-3-methoxycarbonyl-1,2,3,4-tetrahydr
o-.beta.-carboline
This compound was prepared in a manner analogous to the preparation
of Compound 9A. Anal. (c.sub.21,H.sub.22,N.sub.2,O.sub.5) C, H,
N.
1R,3R-1-(3,5-dimethoxy-4-hydroxyphenyl)-3-hydroxymethyl-1,2,3,4-tetrahydro-
.beta.-carboline (Compound 9B)
To a solution of Compound 9a (2.01 g, 5.31 mmol) in 1:1
dioxane/water (20 ml) NaBH.sub.1 (1.00 g, 26.6 mmol) was added, and
the solution was allowed to stir at room temperature for 3 hours.
The solvent was removed under reduced pressure and the remaining
solid was redissolved. The product was precipitated by the addition
of NaCl, and collected by filtration and dried in a vacuum
dessicator to yield 1.43 g (76%) of a white solid which was used
without further purification.
1S,3S-1-(3,5-dimethoxy-4-hydroxyphenyl)-3-hydroxymethyl-1,2,3,4-tetrahydro-
.beta.-carboline
This compound was prepared and analyzed in a manner analogous to
the preparation of Compound 9B.
5R,11aR-3-One-5,4,11,11a-tetrahydro-5-(3,5-dimethoxy-4-hydroxyphenyl)-1H,6H
-oxazolo[3',4':1,6']pyrido[3,4-b]indole (Compound 9)
To a suspension of the amino-alcohol (1.31 g 3.70 mmol) in THF (10
ml), carbonyl diimidazole (1.79 g, 11.1 mmol) was added and the
suspension was allowed to stir for 5 hours. The suspension was
concentrated and 10% NaOH was added. After stirring for an
additional 3 hours, the solution was carefully acidified to pH 6 by
the addition of concentrated HCl and the resulting mixture was
extracted with EtOAc (3.times.50 ml), dried over Na.sub.2 SO.sub.4
and concentrated. .sup.1 NMR (CDCl.sub.3) 7.58(s, 1H), 7.51(d,
J=8.37 Hz, 1H), 7.19(m, 3H), 6.57(s, 2H), 5.52(s, 1H), 5.24(s, 1H),
4.63(t, J=6.65 Hz, 1H), 4.22(m, 2H), 3.83(s, OH), 3.22(m, 1H),
2.92(ddd, J=16.4 Hz, J=10.1 Hz, J=1.8 Hz, 1H). Anal
(C.sub.21,H.sub.20,N.sub.2,O.sub.5) C, H, N.
5S,11aS-3-One-5,4,11,11a-tetrahydro-5-(3,5-dimethoxy-4-hydroxyphenyl)-1H,GH
-oxazolo[3',4':1,6]pyrido[3,4-b]indole (Compound 10)
This compound was prepared in a manner analogous to the preparation
of Compound 9. Anal. (C.sub.21,H.sub.20,N.sub.2,O.sub.5) C, H,
N.
5R,11R,11aS-3-One-11-methoxy-5,4,11,11a-tetrahydro-5-(3,5-dimethoxy-4-hydro
xyphenyl)-1H,6H-oxazolo[3'4':1,6]pyrido[3,4-b]indole (Compound
1C)
To a solution of syringaldehyde dimethyl acetal (0.50 g) and
catalytic p-toluenesulfonic acid in anhydrous CH.sub.2 Cl.sub.2
/MeOH 9:1 (8 ml), Compound 1B was added at 0.degree. C. in small
portions. After stirring for 4 hours the precipitate that formed
was collected by filtering the reaction mixture through a sintered
glass funnel, and was dried in a vacuum desiccator to yield the
pure product in 47% yield. .sup.1 NMR (d.sub.6 -DMSO) 8.42(sb, 1H),
7.65(d, 1H), 7.31(d, 1H), 7.06(m, 2H), 6.48(s, 2H), 5.85(s, 1H),
4.63(d, J=1.8 Hz, 1H), 4.43(m, 3H), 3.64(s, 6H), 3.28(s, 3H).
5R,11R,11aS-3-One-11-hydroxy-5,4,11,11a-tetrahydro-5-(3,5-dimethoxy-4-hydro
xyphenyl)-1H,6H -Oxazolo[3'4:1,6]pyrido[3,4-b]indole (Compound
6)
To a suspension of Compound 1c (1 mmol) in water (8 ml) KIOH (2
mmol) was added, and the solution was brought to reflux and
immediately cooled. The solution was then acidified to ph 7 with
10% HCl, and the precipitate that formed was collected by
filtration in a sintered glass funnel and dried in a vacuum
dessicator.
5R,11R,11aS-3-One-11-methoxy-5,4,11,11a-tetrahydro-5-(3,5-dimethoxy-4-hydro
xyphenyl)-1H,6H-oxazolo[3'4':1,6]pyrido[3,4-b]indole (Compound
1C)
To a solution of syringaldehyde dimethyl acetal (0.50 g) and
catalytic p-toluenesulfonic acid in anhydrous CH.sub.2 Cl.sub.2
/MeOH 9:1 (8 ml), Compound 1B was added at 0.degree. C. in small
portions. After stirring for 4 hours the precipitate that formed
was collected by filtering the reaction mixture through a sintered
glass funnel, and was dried in a vacuum desiccator to yield the
pure product in 47% yield. .sup.1 NMR (d.sub.6 -DMSO) 8.42(sb, 1H),
7.65(d, 1H), 7.31(d, 1H), 7.06(m, 2H), 6.48(s, 2H), 5.85(s, 1H),
4.63(d, J=1.8 Hz, 1H), 4.43(m, 3H), 3.64(s, 6H), 3.28(s, 3H).
5R,11R,11aS-3-One-11-methoxy-5,4,11,11a-tetrahydro-5-(3,5-dimethoxy-4-O-car
bomethoxyphenyl)-1H,6H-oxazolo[3',4':1,6]pyrido[3,4-b]indole
(Compound 1D)
Compound 1C (1.96 g, 4.77 mmol) was suspended in 10 ml of CH.sub.2
Cl.sub.2 and 6.7 ml (47.7 mmol ) TEA. In the suspension, 3.69 ml
(47.7 mmol) of methyl chloroformate was added dropwise at 0.degree.
C., the solution was diluted with CH.sub.2 Cl.sub.2 and washed with
water. The organic fraction was dried over Na.sub.2 SO.sub.4,
filtered and concentrated. Purification by flash chromatography
eluting with 8% acetone in CHCl.sub.3 yielded 2.09 g (94%) of a
white solid. An analytical sample was obtained by recrystallization
from ethyl acetate.
5R,11R,11aS-3-One-11-hydroxy-5,4,11,11a-tetrahydro-5-(3,5-dimethoxy-4-O-car
bomethoxyphenyl)-1H,6H-oxazolo[3',4':1,6]pyrido[3,4-b]indole
(Compound 1E)
To a solution of 1.88 g (7.00 mmol) of compound 1D in a 9:1
dioxane/water solution (30 ml), 130 mg (0.70 mmol) of
paratoluenesulfonic acid (p-TSOH) was added. The solution was
followed to completeion by TLC and diluted with CHCl.sub.3. The
resulting mixture was washed with saturated NaHCO.sub.3, dried over
Na.sub.2 SO.sub.4, filtered and concentrated. Purification by flash
chromatography eluting with 20% acetone in CHCl.sub.3 yielded 1.21
g (66%) of a white solid.
5R,11R,11aS-3-One-11-(4-fluoroanilino)-5,4,11,11a-tetrahydro-5-(3,5-dimetho
xy-4-O-carbomethoxy)-1H,6H-oxazolo[3',4':1,6]pyrido[3,4-b]indole
(Compound 1F)
To a solution of 0.33 g (0.73 mmol) of Compound 1E in anhydrous
dioxane (4 ml), 350 .mu.l (2.5 mmol) of TEA and 160 .mu.l (2.1
mmol) of acetyl chloride were added. After stirring for 15 minutes,
the solvent and unreacted acetyl chloride were removed under
reduced pressure. The reaction vessel was recharged with dioxane,
(diethylene dioxide DDO), 1.4 mmol of 4-fluoro aniline and 1.4 mmol
TEA. The reaction mixture then was heated at 50.degree. C. and
stirred for 6 hours. To this solution, CHCl.sub.3 was added and the
resulting solution was washed with water, dried over Na.sub.2
SO.sub.4, and concentrated. Purification by flash chromatography
eluting with 10% acetone in CHCl.sub.3 yielded 62 mg (16%) of a
white solid.
5R,11R,11aS-3-One-11-(4-fluoroanilino)-5,4,11,11a-tetrahydro-5-(3,5-dimetho
xy-4-hydroxyphenyl)-1H,6H-oxazolo[3',4':1,6]pyrido[3,4-b]indole
(Compound 11)
To a solution of sodium methoxide (90 .mu.mol) in 1 ml methanol,
16.5 mg (30 .mu.mol) of Compound 1F was added and the reaction was
followed to completion by TLC. Saturated NH.sub.4 Cl (200 .mu.l)
was added followed by addition of CH.sub.2 Cl.sub.2. The layers
were separated and the organic layer was dried over Na.sub.2
SO.sub.4, filtered and concentrated. Purification by flash
chromatography eluting with 20% acetone in CHCl.sub.3 yielded 10.8
mg (73%) of a white solid.
Representative syntheses for the aforementioned compounds are
depicted below: ##STR8##
Wherein Ar can be Y, as defined above. ##STR9##
Compounds X, Y and Z have the following structures: ##STR10##
EXAMPLE 2
Induction of DNA Double-Strand Breaks by Azatoxin in the Presence
of HL-60 Nuclear Extract
Drug-induced DNA double-strand breaks were measured first in SV40
DNA in the presence of HL-60 nuclear extracts. SV40 DNA was chosen
because it is a natural substrate of top 2 and is cleaved at many
sites by other top 2 inhibitors. See, for example, Fesen &
Pommier (1991), supra. The smallest azatoxin concentration that
produced detectable cleavage was 5 to 10 .mu.M. Above 10 .mu.M,
cleavage occurred at many sites and was proportional to the
logarithm of azatoxin concentration. The potency of azatoxin was
comparable to that of VP-16 and, as in the case of VP-16,
azatoxin-induced DNA cleavage was not suppressed at high drug
concentrations (up to 1 mM), consistent with azatoxin's not
intercalating into DNA (see below).
EXAMPLE 3
Sequencing of Topoisomerase II Cleavage Sites by Azatoxin
Induction of top 2 cleavage by azatoxin was tested directly with
the use of purified murine leukemia top 2. Since the SV40 nuclear
matrix-associated region has been shown to be cleaved
preferentially by top 2, see Pommier et al. (1991), supra, this
region was chosen for analysis. Sites of cleavage were also
determined by DNA sequencing in the 5' flank of c-myc first intron.
Azatoxin induced many cleavage sites both in the SV40 and the c-myc
DNA fragments. In general, azatoxin induced more cleavage sites
than mitoxantrone, m-AMSA, VM-26 or VP-16. Thus, azatoxin was shown
to be a potent top 2 inhibitor, with a cleavage pattern differing
from those induced by other top 2 inhibitors.
The cleavage pattern of azatoxin also was compared to that of
epipodophyllotoxin derivatives the structures of which seem quite
similar (for drug structures and abbreviations, see table below).
The compound 4'-demethyl-4-desoxypodophyllotoxin (DMDP), with a
structure most similar to azatoxin, induced less cleavage that
azatoxin and at distinct cleavages sites. The .beta.-4-hydroxy
derivative of DMDP, 4'-demethylepipodophyllotoxin (DMEP), was at
least as potent as VP-16, and its cleavage patterns, while similar
to that of VP-16 with some local differences, was different from
that of azatoxin.
EXAMPLE 4
Effects on Topoisomerase Activity
Two different assays were conducted to illuminate the nature of
azatoxin's effects vis-a-vis DNA relaxation. To study the
inhibition of top 2 catalytic activity, topoisomerase reactions
were performed with 0.4 .mu.g native SV40 DNA in 30 .mu.l reaction
buffer for 30 minutes at 37.degree. C. and stopped by adding SDS to
a final concentration of 1% and proteinase K to 400 .mu.g/ml,
followed by incubation for 1 hour at 42.degree. C., essentially as
described by Fesen & Pommier (1989), supra. Agarose gel
electrophoresis was performed in 1% gels made in Tris-Acetate-EDTA
(TAE) buffer (40 mM Tris-Acetate, pH 7.6, 10 mM Na.sub.2 EDTA).
Gels were run at 2 V/cm overnight, washed in water and then stained
with 1 .mu.M ethidiumbromide for 45 minutes. After an additional 45
minutes destaining in 1 mM Mg.sub.2 SO.sub.4, the DNA was
visualized under UV light and photographed with a Polaroid type 57
film.
To assess DNA unwinding, see Pommier et al., Nucleic Acids Res. 15:
6713-31 (1987), the DNA was relaxed first by treatment for 15
minutes with top 1 (20 units), after which the test agent was
added. These steps were carried out at 37.degree. C. DNA-agent-top
1 mixtures were incubated for an additional 30 minutes and then
stopped as described above. Samples were then subjected to agarose
gel electrophoresis as described above.
From the assay data it was determined that azatoxin inhibits top
2-mediated relaxation of native SV40 DNA. At the same time,
azatoxin was observed to produce a substantial amount of linear DNA
without significant increased of nicked DNA.
The DNA unwinding assay, with excess topoisomerase I and relaxed
SV40 DNA, was employed to assess azatoxin intercalation in
accordance with Pommier et al., Nucleic Acids Res. 15: 6713-31
(1987) In fact, azatoxin did not induce detectable DNA unwinding
even at drug concentrations as high as 1 mM. This was also the case
for the 2 azatoxin isomers, 8 and 10, and for the
demethylepipodophyllotoxins, DMDP and DMEP. Similar results were
obtained with supercoiled DNA. The lack of unwinding by azatoxin
strongly indicated that the drug does not intercalate into DNA.
EXAMPLE 5
Effects of Azatoxin Structural Modifications on Topoisomerase II
Inhibition
Three isomers and six azatoxin derivatives, the synthesis of which
is described in Example 1, were tested for drug-induced cleavage
efficiency in the presence either of HL-60 extract or of purified
top 2. The compounds and test results are set out in the table
below.
__________________________________________________________________________
##STR11## ##STR12## ##STR13## ##STR14## ##STR15## Y Stereochemistry
Topoisomerase II COMPOUND STRUCTURE R1 R2 R3 R4 5 11.sup.a
inhibition
__________________________________________________________________________
AZATOXINS Azatoxin 1 OCH3 OH OCH3 H R S +++ 2 1 H OH OCH3 H R S
.cndot. 3 1 H OH H H R S .largecircle. 4 1 OCH3 OCH3 OCH3 H R S
.largecircle. 5 1 H NHSO2CH2 H H R S .largecircle. 6 1 OCH3 OH OCH3
OH R S + 7 4 OCH3 OH OCH3 H R S .largecircle. 8 3 OCH3 OH OCH3 H R
S .largecircle. 9 1 OCH3 OH OCH3 H R R .largecircle. 10 1 OCH3 OH
OCH3 H S S .largecircle. EPIPODOPHYLLOTOXINS DMDP 2 OCH3 OH OCH3 H
R S + DMEP 2 OCH3 OH OCH3 OH R S +++ VP-16 2 OCH3 OH OCH3 .sup.a R
S +++ VM-26 2 OCH3 OH OCH3 .sup.b R S ++++
__________________________________________________________________________
##STR16## ##STR17## - Azatoxin Isomers
The three azatoxin isomers (compounds 8-10 in the table) were found
not to be active as top 2 inhibitors in DNA cleavage assays. The
finding that the two diastereoisomers (9 and 10) were inactive
demonstrated that a strict stereochemical relationship between the
polycyclic ring system and the pendant aromatic ring must exist for
activity. The inactivity of isoazatoxin (8) was surprising and
indicated the great sensitivity of the binding site for these
agents to minor structural modification. Thus, azatoxin and
isoazatoxin (8) differ only in the orientation of the
tetrahydrooxazolopyrido ring fusion into the indole ring; this
change in orientation imparts (1) only a subtle differential
"curve" to the tetracyclic nucleus of the molecule, without
altering the spatial relationship between the indole and phenyl
ring systems, and (2) a change in orientation of the nitrogen
indole.
Azatoxin Derivatives
The results from testing the six azatoxin derivatives for top 2
inhibition also are set out in table. Two of derivatives were
modified on the polycyclic ring system and the others were modified
on the pendant ring.
Hydroxylation at position 11 (R4) of the azatoxin polycyclic ring
system yielded a compound (6) that was structurally similar to DMEP
and that displayed measurable but not strong top 2 activity. This
4-hydroxy substitution differentiates azatoxin from the
4'-demethylpodophyllotoxin framework, since hydroxyl substitution
at this site significantly decreases top 2 activity of azatoxin (1
versus 6) and increases activity of the podophyllotoxins (DMDP,
DMEP). Differing from azatoxin by its polycyclic ring system,
Compound 7 is inactive, again indicating that the structure of the
polycyclic ring system is critical for azatoxin activity.
Modification to the pendant (Y) ring gave the following results.
Monodemethoxylation (2) reduced top 2 activity by a factor 10,
while didemethoxylation (3) abolished the top 2 activity. Position
4' (R2) was also crucial as methylation of the hydroxyl residue (4)
abolished top 2 inhibition. These results are not inconsistent with
those obtained for demethylepipodophyllotoxins. See, for example,
Sinha et al., Eur. J. Cancer 26: 590-93 (1990). Notably, compound
5, in which the azatoxin pendant ring had been placed by that of
AMSA was inactive. These results differentiate the azatoxin family
of top 2 inhibitors from the m-AMSA family of inhibitors.
* * * * *